Low molecular compounds that have a structure similar to the substrate of an enzyme and bind to an active pocket of the enzyme have been used widely as an inhibitor of the enzyme. There is however a plurality of enzymes having a similar active pocket in the living body so that it is known that such low molecular compounds by themselves cannot easily serve as a specific inhibitor. On the other hand, peptidic molecules typified by antibodies have attracted attentions as next-generation pharmaceuticals because they can recognize the surface of a molecule over a wide range and specifically bind to a specific target.
For acquiring such peptidic molecules that bind to a specific target, a method of screening a random peptide library has been used widely. In particular, various in vitro display methods such as ribosome display method and mRNA display method using translation are excellent because a high diversity library can be constructed and screened in a tube in a short period of time. The term “in vitro display method” means a system facilitating concentration and amplification (selection) of active species by linking a phenotype and a genotype coding for the sequence thereof through a non-covalent bond or a covalent bond to display the phenotype on the genotype and using a replication system reconstructed in a test tube. The greatest characteristic of this system is that it is conducted without using a prokaryote or eukaryote as a medium so that a high-activity physiological substance can be isolated from a library having great diversity. As a typical comparison example, phage display using Escherichia coli as a replication medium enables selection from a library having diversity as high as 107. In vitro display, on the other hand, enables searching from a library having diversity as high as 1012. Examples of the in vitro display include ribosome display, mRNA display, and RaPID display (unpublished international patent application PCT/JP2010/68549). As one example, mRNA display will next be described below.
The mRNA display method is a technology of binding a polypeptide to an mRNA which is a template thereof to match the amino acid sequence of the polypeptide to the nucleic acid sequence. By binding puromycin, which is an analogue of the end of an acylated tRNA, to the 3′-end of the mRNA via an appropriate linker and adding it to a translation reaction, puromycin penetrates in the site A of ribosome and forms a covalent bond with a growing peptide. As a result, the peptide molecule which is a translation product is linked to the mRNA via puromycin (Patent Documents 1 to 3, Non-patent Documents 1 and 2).
Thus, the in vitro display enables screening of a peptide library having diversity as high as 1012. Since such peptide library is constructed by making use of a vital function, however, only a peptide library composed only of proteinogenic amino acids has conventionally been constructed. It is expected that if it is possible to overcome the problem of this library composed only of proteinogenic amino acids; incorporate, in an amino acid structure, a low molecular inhibitor having insufficient inhibitory ability or specificity on its own; and construct and screen a library of peptides containing such special amino acid, inhibitors exhibiting high inhibitory ability and selectivity which cannot be attained by using a low molecular compound or a peptide alone can be obtained.
With recent development in technology called “genetic code expansion” or “reprogramming of genetic code”, it actually becomes possible to prepare and screen a library of peptides having a special amino acid by using various display methods such as phage display.
In genetic code expansion, it becomes possible to synthesize proteins or peptides containing a special amino acid by making use of stop codons or artificial four-base codons which are not used for assigning an amino acid in a natural translation system and allocating these codons to the special amino acid. Since the number of stop codons or usable four-base codons is limited, the number of usable special amino acids simultaneously is however limited (substantially, three or less special amino acids).
There are three reports on construction and screening examples of a special peptide library by making use of this “genetic code expansion”. The first one is on the construction of an N-methylphenylalanine-containing peptide library and screening of this library using mRNA display, which is made by R. Roberts, et al (Non-patent Document 3). According to this report, in spite of designing so that N-methylamino acid appears at a certain probability in a random peptide sequence, all the peptides obtained by actually screening with G protein as a target are composed of typical 20 amino acids and an N-methylphenylalanine-containing peptide is not obtained. The second one is on the construction of a peptide library incorporating sulfotyrosine therein and screening of this library using a phage, which is made by P. G. Schultz, et al. According to this report, they have succeeded in constructing a phage which has displayed scFv containing a random region designed so that sulfotyrosine appears at a certain probability and screening the library with gp120, the membrane protein of HIV virus, as a target and thereby actually acquiring scFv that contains sulfotyrosine and binds to gp120 (Non-patent Document 4). When the scFv thus obtained is expressed not in phage display but is expressed as a single substance, however, it becomes insoluble. In addition, it loses activity in the form of Fab so that an antibody having binding ability to a target has not yet been obtained in practice. The third report is on the construction of a library of peptides containing a special amino acid having a boron functional group that binds to saccharide and screening of this library by using a phage, which is also made by P. G. Schultz, et al (Non-patent Document 5). First, by constructing the above-described phage which has displayed scFv containing a random region designed so that a special amino acid appears at a certain probability and then screening the library using a substrate having a saccharide fixed thereon, they have succeeded in acquiring a sequence containing one or two of the special amino acids. There is however no finding that such peptide has specificity to a certain sugar or sugar chain. Also since the boron functional group originally has a property of forming a covalent bond with the hydroxyl group of sugar, the possibility of nonspecific binding between them cannot be denied. In short, a technology of acquiring scFv having a biologically significant peptide sequence that binds to a sugar chain or sugar protein has not yet been developed.
Any of the above-described technologies is limited to the construction of a library containing only one special amino acid and has not succeeded in acquiring a peptide containing desired physiological functions. They are therefore crude technologies from the standpoint of versatility and reliability of the technology.
Since the 2000s, “genetic code reprogramming” (reprogramming by initialization) in which a special amino acid is assigned to an vacant codon generated by removing a natural amino acid from the system has been developed, making it possible to use four or more special amino acids (Non-patent Documents 6 to 8). No examples have however been known yet in which a random peptide library containing a plurality of special amino acids is constructed by making use of genetic code reprogramming and a peptidic molecule that binds to a specific target is searched from the library.